Increase In Cytoplasmic Free Ca2 Research Articles

Ca2+ and force during dynamic contractions in mouse intact skeletal muscle fibers

Muscle fiber force production is determined by the excitation frequency of motor nerves, which induce transient increases in cytoplasmic free Ca2+ concentration ([Ca2+]i) and the force-generating capacity of the actomyosin cross-bridges. Previous studies suggest that, in addition to altered cross-bridge properties, force changes during dynamic (concentric or eccentric) contraction might be affected by Ca2+-dependent components. Here we investigated this by measuring [Ca2+]i and force in mouse muscle fibers undergoing isometric, concentric, and eccentric contractions. Intact single muscle fibers were dissected from the flexor digitorum brevis muscle of mice. Fibers were electrically activated isometrically at 30–100 Hz and after reaching the isometric force plateau, they were actively shortened or stretched. We calculated the ratio (relative changes) in force and [Ca2+]i attained in submaximal (30 Hz) and near-maximal (100 Hz) contractions under isometric or dynamic conditions. Tetanic [Ca2+]i was similar during isometric, concentric and eccentric phases of contraction at given stimulation frequencies while the forces were clearly different depending on the contraction types. The 30/100 Hz force ratio was significantly lower in the concentric (44.1 ± 20.3%) than in the isometric (50.3 ± 20.4%) condition (p = 0.005), whereas this ratio did not differ between eccentric and isometric conditions (p = 0.186). We conclude that the larger force decrease by decreasing the stimulation frequency during concentric than during isometric contraction is caused by decreased myofibrillar Ca2+ sensitivity, not by the decreased [Ca2+]i.

Total Calcium Content of Sarcoplasmic Reticulum and Mitochondria in Ryanodine Receptor Variant Muscle

Ryanodine receptor (RyR) mutations can induce susceptibility to malignant hyperthermia or myopathies. The mutation of the RyR can cause an increased leak of Ca2+ from the sarcoplasmic reticulum (SR). The leak of SR Ca2+ is associated with an increase in cytoplasmic free Ca2+, which may activate cytoplasmic proteases or potentially enter the mitochondria to increase the load of calcium inside this organelle. A major gap in our understanding the issues associated with RyR variants is the effect on Ca2+-handling by the cellular organelles, which we could better address if we had information on the total calcium content of the SR and mitochondria under conditions of known RyR mutations and associated Ca2+ leak. We determined the RyR Ca2+ leak in extensor digitorum longus muscle fibres of wild-type (WT), heterozygous (HET) and homozygous (HOM) mice for the RyR1 mutant p.G2435R (Lopez et al 2018, BJA) using recently developed techniques (Cully et al 2018, PNAS). We observed an increasing leak with increasing number of mutated RyR alleles, from WT to HET to HOM mice. Next, we used a membrane-lysis method to release all the total compartmentalized calcium onto the contractile apparatus to use the ensuing force response to determine SR calcium content (Fryer & Stephenson, 1996. J.Physiol; Lamboley et al 2013, J.Physiol). We modified this technique to isolate the depletion of mitochondrial calcium to allow determination of total calcium within this compartment. Our results show a progressive decrease in the SR total calcium content with increasing RyR Ca2+ leak. The mitochondrial total calcium content increased significantly from WT, to that in the HET, to that in the HOM mice.

Peucedanum japonicum Thunb. ethanol extract suppresses RANKL-mediated osteoclastogenesis.

The constituents of Peucedanum japonicum Thunb. (PJ) exhibit biological and pharmacological activities, including anti-obesity, anti-oxidant and anti-allergic activities. The aim of the present study was to examine in vitro effects of PJ in RANKL-induced signaling pathways, which determine osteoclast differentiation. PJ ethanol extract (PEE) exhibited anti-osteoporotic activity by disrupting the phospholipase C (PLC)-Ca2+-c-Fos/cAMP response element-binding protein (CREB)-nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway during osteoclastogenesis. Murine bone marrow-derived macrophages (BMMs) were cultured and used to determine the effects of PJ in the receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclastogenesis. The effects of PEE in the RANKL-mediated signaling cascade were evaluated using a standard in vitro osteoclastogenesis system. PEE treatment of BMMs significantly reduced the number of RANKL-mediated tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells (P<0.05 for 5 and 10 µg/ml PEE, P<0.01 for 25 and 50 µg/ml PEE), without cytotoxic effects. Furthermore, the expression of differentiation-related marker genes, including TRAP, Oscar, Cathepsin K, dendrocyte expressed seven transmembrane protein, ATPase H+ Transporting V0 Subunit D2 and NFATc1, were markedly suppressed. PEE induced a transient increase in free cytoplasmic Ca2+ ([Ca2+]i) mobilization via voltage-gated Ca2+ channels and PLC-sensitive pathways. Transient [Ca2+]i increase consequently resulted in the suppression of c-Fos, CREB and NFATc1 activities. These findings highlight the potential use of PJ in treating bone disorders caused by osteoclast overgrowth.

A novel approach to regulate cell membrane permeability for ATP and NADH formation in Saccharomyces cerevisiae induced by air cold plasma**Supported by National Natural Science Foundation of China (Nos. 21246012, 21306015 and 21476032).

Air cold plasma has been used as a novel method for enhancing microbial fermentation. The aim of this work was to explore the effect of plasma on membrane permeability and the formation of ATP and NADH in Saccharomyces cerevisiae, so as to provide valuable information for large-scale application of plasma in the fermentation industry. Suspensions of S. cerevisiae cells were exposed to air cold plasma for 0, 1, 2, 3, 4 and 5 min, and then subjected to various analyses prior to fermentation (0 h) and at the 9 and 21 h stages of fermentation. Compared with non-exposed cells, cells exposed to plasma for 1 min exhibited a marked increase in cytoplasmic free Ca2+ concentration as a result of the significant increase in membrane potential prior to fermentation. At the same time, the ATP level in the cell suspension decreased by about 40%, resulting in a reduction of about 60% in NADH prior to culturing. However, the levels of ATP and NADH in the culture at the 9 and 21 h fermentation stages were different from the level at 0 h. Taken together, the results indicated that exposure of S. cerevisiae to air cold plasma could increase its cytoplasmic free Ca2+ concentration by improving the cell membrane potential, consequently leading to changes in ATP and NADH levels.

Echinacoside Inhibits Glutamate Release by Suppressing Voltage-Dependent Ca(2+) Entry and Protein Kinase C in Rat Cerebrocortical Nerve Terminals.

The glutamatergic system may be involved in the effects of neuroprotectant therapies. Echinacoside, a phenylethanoid glycoside extracted from the medicinal Chinese herb Herba Cistanche, has neuroprotective effects. This study investigated the effects of echinacoside on 4-aminopyridine-evoked glutamate release in rat cerebrocortical nerve terminals (synaptosomes). Echinacoside inhibited Ca2+-dependent, but not Ca2+-independent, 4-aminopyridine-evoked glutamate release in a concentration-dependent manner. Echinacoside also reduced the 4-aminopyridine-evoked increase in cytoplasmic free Ca2+ concentration but did not alter the synaptosomal membrane potential. The inhibitory effect of echinacoside on 4-aminopyridine-evoked glutamate release was prevented by ω-conotoxin MVIIC, a wide-spectrum blocker of Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels, but was insensitive to the intracellular Ca2+ release-inhibitors dantrolene and 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157). Furthermore, echinacoside decreased the 4-aminopyridine-induced phosphorylation of protein kinase C, and protein kinase C inhibitors abolished the effect of echinacoside on glutamate release. According to these results, we suggest that the inhibitory effect of echinacoside on evoked glutamate release is associated with reduced voltage-dependent Ca2+ entry and subsequent suppression of protein kinase C activity.

Aged Garlic Extract Inhibits Human Platelet Aggregation by Altering Intracellular Signaling and Platelet Shape Change

Background:Increased platelet aggregation plays a pivotal role in the etiology of cardiovascular disease. Upon platelet aggregation, an increase in free cytoplasmic Ca2+ results in the inhibition of soluble guanylyl cyclase (sGC) and adenylyl cyclase (AC), leading to a decrease in cyclic guaninosine-5′-monophosphate (cGMP) and cAMP, respectively. This leads to the activation of the glycoprotein IIb/IIIa (GPIIb/IIIa) fibrinogen receptor, resulting in platelet shape change. Aged garlic extract (AGE) decreases platelet aggregation; however, the mechanisms involved are not clearly defined.Objective:Our objective was to investigate the effects of AGE on intraplatelet cell signaling and platelet shape change.Methods:Platelets from 14 participants were studied. Platelet aggregation was induced by ADP in the presence of AGE up to a concentration of 6.25% (vol:vol) alone or in combination with 3-morpholinosydnonimine (Sin-1), a nitric oxide donor. The experiments with AGE were repeated in the presence of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. In a series of separate experiments, platelet aggregation was induced in the presence of either 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an sGC inhibitor, or 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536), an AC inhibitor, or a combination of both in the presence of IBMX and AGE. Intraplatelet cGMP and cAMP were measured. The platelets were also subjected to scanning electron microscopic analysis, and their binding to fibrinogen was determined.Results:AGE decreased platelet aggregation at all concentrations tested; this decrease was more marked in the presence of Sin-1 and ranged between 15% and 67%.The presence of IBMX also led to a decrease (17–35%) in platelet aggregation at all AGE concentrations and a significant decrease in the amounts of cGMP (24–41%) and cAMP (19–70%), respectively, in the presence of ODQ and SQ22536. The presence of AGE significantly inhibited the binding of activated platelets to fibrinogen, preventing changes in platelet shape.Conclusion:These results indicate that AGE inhibits platelet aggregation by increasing cyclic nucleotides and inhibiting fibrinogen binding and platelet shape change.

Ion channels contribute to the regulation of cell sheet forces during Drosophila dorsal closure

We demonstrate that ion channels contribute to the regulation of dorsal closure in Drosophila, a model system for cell sheet morphogenesis. We find that Ca2+ is sufficient to cause cell contraction in dorsal closure tissues, as UV-mediated release of caged Ca2+ leads to cell contraction. Furthermore, endogenous Ca2+ fluxes correlate with cell contraction in the amnioserosa during closure, whereas the chelation of Ca2+ slows closure. Microinjection of high concentrations of the peptide GsMTx4, which is a specific modulator of mechanically gated ion channel function, causes increases in cytoplasmic free Ca2+ and actomyosin contractility and, in the long term, blocks closure in a dose-dependent manner. We identify two channel subunits, ripped pocket and dtrpA1 (TrpA1), that play a role in closure and other morphogenetic events. Blocking channels leads to defects in force generation via failure of actomyosin structures, and impairs the ability of tissues to regulate forces in response to laser microsurgery. Our results point to a key role for ion channels in closure, and suggest a mechanism for the coordination of force-producing cell behaviors across the embryo.

Ion channels contribute to the regulation of cell sheet forces during Drosophila dorsal closure

We demonstrate that ion channels contribute to the regulation of dorsal closure in Drosophila, a model system for cell sheet morphogenesis. We find that Ca(2+) is sufficient to cause cell contraction in dorsal closure tissues, as UV-mediated release of caged Ca(2+) leads to cell contraction. Furthermore, endogenous Ca(2+) fluxes correlate with cell contraction in the amnioserosa during closure, whereas the chelation of Ca(2+) slows closure. Microinjection of high concentrations of the peptide GsMTx4, which is a specific modulator of mechanically gated ion channel function, causes increases in cytoplasmic free Ca(2+) and actomyosin contractility and, in the long term, blocks closure in a dose-dependent manner. We identify two channel subunits, ripped pocket and dtrpA1 (TrpA1), that play a role in closure and other morphogenetic events. Blocking channels leads to defects in force generation via failure of actomyosin structures, and impairs the ability of tissues to regulate forces in response to laser microsurgery. Our results point to a key role for ion channels in closure, and suggest a mechanism for the coordination of force-producing cell behaviors across the embryo.

Cytoplasmic free Ca2+ is essential for multiple steps in malaria parasite egress from infected erythrocytes

BackgroundEgress of Plasmodium falciparum, from erythrocytes at the end of its asexual cycle and subsequent parasite invasion into new host cells, is responsible for parasite dissemination in the human body. The egress pathway is emerging as a coordinated multistep programme that extends in time for tens of minutes, ending with rapid parasite extrusion from erythrocytes. While the Ca2+ regulation of the invasion of P. falciparum in erythrocytes is well established, the role of Ca2+ in parasite egress is poorly understood. This study analysed the involvement of cytoplasmic free Ca2+ in infected erythrocytes during the multistep egress programme of malaria parasites.MethodsLive-cell fluorescence microscopy was used to image parasite egress from infected erythrocytes, assessing the effect of drugs modulating Ca2+ homeostasis on the egress programme.ResultsA steady increase in cytoplasmic free Ca2+ is found to precede parasite egress. This increase is independent of extracellular Ca2+ for at least the last two hours of the cycle, but is dependent upon Ca2+ release from internal stores. Intracellular BAPTA chelation of Ca2+ within the last 45 minutes of the cycle inhibits egress prior to parasitophorous vacuole swelling and erythrocyte membrane poration, two characteristic morphological transformations preceding parasite egress. Inhibitors of the parasite endoplasmic reticulum (ER) Ca2+-ATPase accelerate parasite egress, indicating that Ca2+ stores within the ER are sufficient in supporting egress. Markedly accelerated egress of apparently viable parasites was achieved in mature schizonts using Ca2+ ionophore A23187. Ionophore treatment overcomes the BAPTA-induced block of parasite egress, confirming that free Ca2+ is essential in egress initiation. Ionophore treatment of immature schizonts had an adverse effect inducing parasitophorous vacuole swelling and killing the parasites within the host cell.ConclusionsThe parasite egress programme requires intracellular free Ca2+ for egress initiation, vacuole swelling, and host cell cytoskeleton digestion. The evidence that parasitophorous vacuole swelling, a stage of unaffected egress, is dependent upon a rise in intracellular Ca2+ suggests a mechanism for ionophore-inducible egress and a new target for Ca2+ in the programme liberating parasites from the host cell. A regulatory pathway for egress that depends upon increases in intracellular free Ca2+ is proposed.

Molecular Determinants of Cav1.2 Calcium Channel Inactivation.

Voltage-gated L-type Cav1.2 calcium channels couple membrane depolarization to transient increase in cytoplasmic free Ca2+ concentration that initiates a number of essential cellular functions including cardiac and vascular muscle contraction, gene expression, neuronal plasticity, and exocytosis. Inactivation or spontaneous termination of the calcium current through Cav1.2 is a critical step in regulation of these processes. The pathophysiological significance of this process is manifested in hypertension, heart failure, arrhythmia, and a number of other diseases where acceleration of the calcium current decay should present a benefit function. The central issue of this paper is the inactivation of the Cav1.2 calcium channel mediated by multiple determinants.

Decreased Liver Tumorigenesis in Sulfatase 2 (SULF2) Knockout Mice May Be Mediated Through Inhibition of the Epithelial-Mesenchymal Transition (EMT)

Background and Aim. The pathogenesis and progression of pancreatic cancer involves conversion of TGFβ suppressive signaling in early lesions to an unmasked, TGFβ proliferative signaling cascade in metastatic lesions. We have previously demonstrated that TGFβ proliferative signaling downregulates the tumor suppressor PTEN and induces motility of pancreatic cancer cells after ligand-receptor binding with subsequent increase in cytoplasmic free Ca2+ concentration ([Ca2+]cyt) and activation of PKCα and NF-κB. [Ca2+]cyt homeostasis is important to maintain cell functionality dysregulation of which contributes to tumorigenesis of pancreas. Transient receptor potential ion channels (TRPC) help regulate [Ca2+]cyt, and here we evaluated the role of TRPC channels in regulating TGFβ proliferative signaling. Methods. BxPc3 pancreatic cancer cells were treated with TGFβ (10 ng/mL), and TRPC and PTEN expression were analyzed by RT-PCR and/or Western blots. [Ca2+]cyt was assessed using Fura-2 fluorescence ratio digital imaging. Pharmacological reagents (TRPC inhibitors: 2-APB, SKF96365, and LaCl3) and siRNAs to TRPCs were used to determine the role of TRPCs on TGFβ-induced PTEN regulation and cell migration. Cell motility was assessed by Boyden chamber migration assay. Results. BxPc3 cells express TRPC1, 4, and 6 isoforms. TGFβ induced an increase in [Ca2+]cyt in Ca2+-free solutions and also induced an increase in [Ca2+]cyt when extracellular Ca2+ was restored. TGFβ-induced Ca2+ entry involves the activation of TRPC, as the TRPC inhibitors 2-APB, SKF96365, and LaCl3, were able to reduce peak [Ca2+]cyt by 80%, 40%, and 29%, respectively, and reversed TGFβ-induced PTEN downregulation. The 2-APB, SKF96365, and LaCl3 inhibitors also reduced TGFβinduced cell motility. Using siRNA, inhibition of TRPC1 but not other isoforms, successfully prevented TGFβ-induced cell motility. Conclusion. TRPC1 regulates TGFβ-induced downregulation of PTEN and pancreatic cell motility. This aspect of TGFβ regulation of PTEN and pancreatic cell motility might be exploited therapeutically to control pancreatic cancer metastasis.

Clinical Outcome of Polypoid Lesion of Gallbladder 10 mm or Larger

Background and Aim. The pathogenesis and progression of pancreatic cancer involves conversion of TGFβ suppressive signaling in early lesions to an unmasked, TGFβ proliferative signaling cascade in metastatic lesions. We have previously demonstrated that TGFβ proliferative signaling downregulates the tumor suppressor PTEN and induces motility of pancreatic cancer cells after ligand-receptor binding with subsequent increase in cytoplasmic free Ca2+ concentration ([Ca2+]cyt) and activation of PKCα and NF-κB. [Ca2+]cyt homeostasis is important to maintain cell functionality dysregulation of which contributes to tumorigenesis of pancreas. Transient receptor potential ion channels (TRPC) help regulate [Ca2+]cyt, and here we evaluated the role of TRPC channels in regulating TGFβ proliferative signaling. Methods. BxPc3 pancreatic cancer cells were treated with TGFβ (10 ng/mL), and TRPC and PTEN expression were analyzed by RT-PCR and/or Western blots. [Ca2+]cyt was assessed using Fura-2 fluorescence ratio digital imaging. Pharmacological reagents (TRPC inhibitors: 2-APB, SKF96365, and LaCl3) and siRNAs to TRPCs were used to determine the role of TRPCs on TGFβ-induced PTEN regulation and cell migration. Cell motility was assessed by Boyden chamber migration assay. Results. BxPc3 cells express TRPC1, 4, and 6 isoforms. TGFβ induced an increase in [Ca2+]cyt in Ca2+-free solutions and also induced an increase in [Ca2+]cyt when extracellular Ca2+ was restored. TGFβ-induced Ca2+ entry involves the activation of TRPC, as the TRPC inhibitors 2-APB, SKF96365, and LaCl3, were able to reduce peak [Ca2+]cyt by 80%, 40%, and 29%, respectively, and reversed TGFβ-induced PTEN downregulation. The 2-APB, SKF96365, and LaCl3 inhibitors also reduced TGFβinduced cell motility. Using siRNA, inhibition of TRPC1 but not other isoforms, successfully prevented TGFβ-induced cell motility. Conclusion. TRPC1 regulates TGFβ-induced downregulation of PTEN and pancreatic cell motility. This aspect of TGFβ regulation of PTEN and pancreatic cell motility might be exploited therapeutically to control pancreatic cancer metastasis.

Targeting Hypertension With a New Adenosine Triphosphate–sensitive Potassium Channel Opener Iptakalim

Hypertension is the most common cardiovascular disease. The discovery of the antihypertensive action of adenosine triphosphate-sensitive potassium (K(ATP)) channel openers was a significant advance in the treatment of hypertension. Iptakalim is a novel K(ATP) channel opener with a unique chemical structure that differs from other K(ATP) openers. Among the 3 different subtypes of K(ATP) channels heterologously expressed in human embryonic kidney cells and Xenopus oocytes, iptakalim exhibits significant selectivity for SUR2B/Kir6.1 channels, mild effects on SUR2A/Kir6.2 channels, and fails to open SUR1/Kir6.2 channels. Iptakalim is a more potent activator of the SUR2B/Kir6.1 subtype of K(ATP) channels than diazoxide and pinacidil, the 2 most commonly studied K(ATP) channel openers. Iptakalim selectively produces arteriolar vasodilation with essentially no effect on the capacitance vessels. It can preferentially relax arterioles and small arteries, without affecting large arteries. Furthermore, iptakalim strongly lowers the blood pressure of hypertensive rodents and humans but has little effect on normotensive rodents and humans. Selective antihypertensive action is not observed with pinacidil or diazoxide and may be due to the high selectivity of iptakalim for the SUR2B/Kir6.1 subtype of K(ATP) channels, as well as its selective relaxation of resistance vessels. In pulmonary arterial smooth muscle cells, iptakalim inhibits the increase of cytoplasmic free Ca2+ concentration, as well as cell proliferation induced by endothelin-1. Furthermore, iptakalim has exerted protective effects against hypertensive damage to target organs in rats and improves endothelial dysfunction associated with cardiovascular diseases by selective activation of the SUR2B/Kir6.1 subtype of K(ATP) channels expressed in the endothelium. Clinical trials of iptakalim in the treatment of mild-moderate hypertension have been completed in China. In additional to strong antihypertensive efficacy, iptakalim seems to have a favorable safety and tolerability profile. Iptakalim is a promising new generation antihypertensive drug.

Pancreatitis: the neglected duct

Acute and chronic pancreatitis are multifactorial diseases that involve inflammation of the pancreas, generation of toxic mediators and mistargeting of digestive enzymes that eventually result in destruction of the parenchyma.1...

Estradiol regulation of progesterone synthesis in the brain

Steroidogenesis is now recognized as a global phenomenon in the brain, but how it is regulated and its relationship to circulating steroids of peripheral origin have remained more elusive issues. Neurosteroids, steroids synthesized de novo in nervous tissue, have a large range of actions in the brain, but it is only recently that the role of neuroprogesterone in the regulation of arguably the quintessential steroid-dependent neural activity, regulation of the reproduction has been appreciated. Circuits involved in controlling the LH surge and sexual behaviors were thought to be influenced by estradiol and progesterone synthesized in the ovary and perhaps the adrenal. It is now apparent that estradiol of ovarian origin regulates the synthesis of neuroprogesterone, and it is the locally produced neuroprogesterone that is involved in the initiation of the LH surge and subsequent ovulation. In this model, estradiol induces the transcription of progesterone receptors while stimulating synthesis of neuroprogesterone. Although the complete signaling cascade has not been elucidated, many of the features have been characterized. The synthesis of neuroprogesterone occurs primarily in astrocytes and requires the interaction of membrane-associated estrogen receptor-alpha with metabotropic glutamate receptor-1a. This G protein-coupled receptor activates a phospholipase C that in turn increases inositol trisphosphate (IP3) levels mediating the release of intracellular stores of Ca2+ via an IP3 receptor gated Ca2+ channel. The large increase in free cytoplasmic Ca2+ ([Ca2+]i) stimulates the synthesis of progesterone, which can then diffuse out of the astrocyte and activate estradiol-induced progesterone receptors in local neurons to trigger the neural cascade to produce the LH surge. Thus, it is a cooperative action of astrocytes and neurons that is needed for estrogen positive feedback and stimulation of the LH surge.

Protein-tyrosine kinase CAKβ/PYK2 is activated by binding Ca2+/calmodulin to FERM F2 α2 helix and thus forming its dimer

CAKbeta (cell adhesion kinase beta)/PYK2 (proline-rich tyrosine kinase 2) is the second protein-tyrosine kinase of the FAK (focal adhesion kinase) subfamily. It is different from FAK in that it is activated following an increase in cytoplasmic free Ca2+. In the present study we have investigated how Ca2+ activates CAKbeta/PYK2. Calmodulin-agarose bound CAKbeta/PYK2, but not FAK, in the presence of CaCl2. An alpha-helix (F2-alpha2) present in the FERM (band four-point-one, ezrin, radixin, moesin homology) F2 subdomain of CAKbeta/PYK2 was the binding site of Ca2+/calmodulin; a mutant of this region, L176A/Q177A (LQ/AA) CAKbeta/PYK2, bound to Ca2+/calmodulin much less than the wild-type. CAKbeta/PYK2 is known to be prominently tyrosine phosphorylated when overexpressed from cDNA. The enhanced tyrosine phosphorylation was inhibited by W7, an inhibitor of calmodulin, and by a cell-permeable Ca2+ chelator and was almost defective in the LQ/AA-mutant CAKbeta/PYK2. CAKbeta/PYK2 formed a homodimer on binding of Ca2+/calmodulin, which might then induce a conformational change of the kinase, resulting in transphosphorylation within the dimer. The dimer was formed at a free-Ca2+ concentration of 8-12 muM and was stable at 500 nM Ca2+, but dissociated to a monomer in a Ca2+-free buffer. The dimer formation of CAKbeta/PYK2 FERM domain was partially defective in the LQ/AA-mutant FERM domain and was blocked by W7 and by a synthetic peptide with amino acids 168-188 of CAKbeta/PYK2, but not by a peptide with its LQ/AA-mutant sequence. It is known that the F2-alpha2 helix is found immediately adjacent to a hydrophobic pocket in the FERM F2 lobe, which locks, in the autoinhibited FAK, the C-lobe of the kinase domain. Our results indicate that Ca2+/calmodulin binding to the FERM F2-alpha2 helix of CAKbeta/PYK2 releases its kinase domain from autoinhibition by forming a dimer.

Mg2+Deprivation Elicits Rapid Ca2+Uptake and Activates Ca2+/Calcineurin Signaling inSaccharomyces cerevisiae

To learn about the cellular processes involved in Mg(2+) homeostasis and the mechanisms allowing cells to cope with low Mg(2+) availability, we performed RNA expression-profiling experiments and followed changes in gene activity upon Mg(2+) depletion on a genome-wide scale. A striking portion of genes up-regulated under Mg(2+) depletion are also induced by high Ca(2+) and/or alkalinization. Among the genes significantly up-regulated by Mg(2+) starvation, Ca(2+) stress, and alkalinization are ENA1 (encoding a P-type ATPase sodium pump) and PHO89 (encoding a sodium/phosphate cotransporter). We show that up-regulation of these genes is dependent on the calcineurin/Crz1p (calcineurin-responsive zinc finger protein) signaling pathway. Similarly to Ca(2+) stress, Mg(2+) starvation induces translocation of the transcription factor Crz1p from the cytoplasm into the nucleus. The up-regulation of ENA1 and PHO89 upon Mg(2+) starvation depends on extracellular Ca(2+). Using fluorescence resonance energy transfer microscopy, we demonstrate that removal of Mg(2+) results in an immediate increase in free cytoplasmic Ca(2+). This effect is dependent on external Ca(2+). The results presented indicate that Mg(2+) depletion in yeast cells leads to enhanced cellular Ca(2+) concentrations, which activate the Crz1p/calcineurin pathway. We provide evidence that calcineurin/Crz1p signaling is crucial for yeast cells to cope with Mg(2+) depletion stress.

Arachidonic acid enhances intracellular [Ca2+]i increase and mitochondrial depolarization induced by glutamate in cerebellar granule cells

Maturation of primary neuronal cultures is accompanied by an increase in the proportion of cells that exhibit biphasic increase in free cytoplasmic Ca2+ ([Ca2+]i) followed by synchronic decrease in electrical potential difference across the inner mitochondrial membrane (DeltaPsim) in response to stimulation of glutamate receptors. In the present study we have examined whether the appearance of the second phase of [Ca2+]i change can be attributed to arachidonic acid (AA) release in response to the effect of glutamate (Glu) on neurons. Using primary culture of rat cerebellar granule cells we have investigated the effect of AA (1-20 microM) on [Ca2+]i, DeltaPsim, and [ATP] and changes in these parameters induced by neurotoxic concentrations of Glu (100 microM, 10-40 min). At =10 microM, AA caused insignificant decrease in DeltaPsim without any influence on [Ca2+]i. The mitochondrial ATPase inhibitor oligomycin enhanced AA-induced decrease in DeltaPsim; this suggests that AA may inhibit mitochondrial respiration. Addition of AA during the treatment with Glu resulted in more pronounced augmentation of [Ca2+]i and the decrease in DeltaPsim than the changes in these parameters observed during independent action of AA; removal of Glu did not abolish these changes. An inhibitor of the cyclooxygenase and lipoxygenase pathways of AA metabolism, 5,8,11,14-eicosatetraynoic acid, increased the proportion of neurons characterized by Glu-induced biphasic increase in [Ca2+]i and the decrease in DeltaPsim. Palmitic acid (30 microM) did not increase the percentage of neurons exhibiting biphasic response to Glu. Co-administration of AA and Glu caused 2-3 times more pronounced decrease in ATP concentrations than that observed during the independent effect of AA and Glu. The data suggest that AA may influence the functional state of mitochondria, and these changes may promote biphasic [Ca2+]i and DeltaPsim responses of neurons to the neurotoxic effect of Glu.

Discovery of modulators of B cell receptor response using a calcium mobilization assay.

The B cell receptor (BCR) induces a sustained increase in cytoplasmic free Ca2+ upon activation. This ensures the progression of calcium-dependent signaling processes such as protein phosphorylation, gene transcription, cell cycle progression, or apoptosis. In an effort to identify and characterize potential immunomodulatory agents we have used freshly isolated and Ramos B cells to establish a FLIPR-based method for measuring effects of various agents on BCR-mediated Ca2+ signaling. Here we report on a Ca2+ assay that allows sensitive and robust detection of inhibitory actions of both mAbs as well as inhibitors of downstream tyrosine kinase targets. The observed effects on Ca2+ signaling have been corroborated with FACS analyses of develomental and proliferative functions. Our method can be applied to the systematic evaluation of potential drugs which may modify B cell development, activation or proliferation through effects on Ca2+ signaling.

Antifungal effects and mechanism of action of viscotoxin A3

Viscotoxins are cationic proteins, isolated from different mistletoe species, that belong to the group of thionins, a group of basic cysteine-rich peptides of approximately 5 kDa. They have been shown to be cytotoxic to different types of cell, including animal, bacterial and fungal. The aim of this study was to obtain information on the cell targets and the mechanism of action of viscotoxin isoform A3 (VtA3). We describe a detailed study of viscotoxin interaction with fungal-derived model membranes, its location inside spores of Fusarium solani, as well as their induced spore death. We show that VtA3 induces the appearance of ion-channel-like activity, the generation of H2O2, and an increase in cytoplasmic free Ca2+. Moreover, we show that Ca2+ is involved in VtA3-induced spore death and increased H2O2 concentration. The data presented here strongly support the notion that the antifungal activity of VtA3 is due to membrane binding and channel formation, leading to destabilization and disruption of the plasma membrane, thereby supporting a direct role for viscotoxins in the plant defence mechanism.